The present invention relates to a bacterium of the genus Lactobacillus with desirable characteristics suitable for use in a vaginal medicant. More particularly, this invention relates to a vaginal medicant having a substantially pure culture of preserved microbial cells, to a preservation matrix, to a method for preserving the microbial cells within the preservation matrix, and to methods for preventing and treating vaginal infections.
Lower genital tract infections, including sexually transmitted diseases (STDs) are some of the most common clinical problems among women of childbearing age. Over 10 million office visits each year in the United States are attributed to vaginal complaints. Vaginal discharge can be due to vaginal infections (yeast, bacterial vaginosis and trichomonas) or cervical infections (gonorrhea or chlamydia). Additionally, there is a body of evidence linking vaginal infections to preterm delivery, low birth weight, and neonatal mortality, which are some of the most important problems faced in obstetrics. Bacterial vaginosis is one of the most common genital infections in pregnancy. Women with bacterial vaginosis diagnosed during the second trimester of pregnancy are 40 percent more likely to give birth to a premature, low-birth weight infant than women without bacterial vaginosis. The prevention of even a small proportion of such births could translate into large monetary savings and a decrease in neonatal morbidity and mortality.
Lactobacilli are gram positive rods that are a part of the microbial flora of the human gut, mouth, and vagina. Vaginal Lactobacilli are thought to play an important role in resistance to infection via production of lactic acid and acidification of the vagina or by production of other antimicrobial products, such as hydrogen peroxide H2O2. It has been demonstrated that women with predominant vaginal Lactobacillus flora have a 50% lower frequency of gonorrhea, chlamydial infections, trichomoniasis and bacterial vaginosis. The presence of H2O2-producing Lactobacilli in the vagina have been linked to a decreased frequency of bacterial vaginosis, symptomatic yeast vaginitis and sexually transmitted pathogens including Neisseria gonorrhea, Chlamydia trachomatis, and Trichomonas vaginalis. In vitro studies have demonstrated that H2O2-producing Lactobacilli have potent bactericidal and viricidal properties against vaginal pathogens and even against human immunodeficiency virus (HIV).
Unfortunately, many women of childbearing age lack vaginal Lactobacilli. The vaginal ecosystem is dynamically affected by medications, general health status, sexual practices and contraception. Many vaginal and systemic medications may kill vaginal Lactobacilli. Hence, treatment of sexually transmitted diseases with antibiotics may place women at increased risk for repeated acquisition of the diseases. These findings, along with the widespread belief that Lactobacilli generally promote vaginal health, have suggested to clinicians that women should recolonize the vagina with Lactobacillus to prevent or treat genital tract infections.
Lactobacillus products for intravaginal or oral use have been available for over 100 years in the form of xe2x80x9cacidophilusxe2x80x9d preparations available in health food stores, and acidophilus milk or yogurt bought in grocery stores (e.g., these products typically advertise the inclusion of a strain of Lactobacillus acidophilus). These products have included vaginal suppositories containing lyophilized Lactobacillus acidophilus of human origin as well as various nutritional supplements. These products have been largely non-efficacious due to the failure of the products to colonize the vagina with the exogenous Lactobacilli. These failures are likely due to the poor quality of the commercially available products. It has been documented that Lactobacillus products sold as part of foods or as Lactobacillus supplements are often contaminated with other potential pathogens. In addition, Lactobacillus obtained from yogurt has been shown to be unable to bind to vaginal epithelial cells. The binding of Lactobacilli to the epithelial cells is a necessary step to establish colonization of the host organism. Therefore, the use of commercially available Lactobacillus products is thought to have little utility in prevention or treatment of vaginal infection because the products contain inappropriate microbe strains (e.g., many contain strains which are rarely recovered from the vagina), are contaminated with other potentially pathogenic organisms, have low microbe viability, and/or the microbes typically do not have the ability to bind to vaginal epithelial cells and establish colonization.
Therefore, there is a need for a product for the treatment of vaginal infections which can be manufactured under exacting conditions and which uses appropriate human strains of Lactobacillus having in vivo microbicidal properties, active adherence and an effective potency of viable microbes.
The present invention generally relates to a bacterium of the genus Lactobacillus with desirable characteristics suitable for use in a vaginal medicant. More particularly, this invention relates to vaginal, rectal and oral medicants having a substantially pure culture of preserved microbial cells, to a preservation matrix and to methods for preventing and treating vaginal infections.
The unique strains of Lactobacillus disclosed herein, when administered in vivo, will colonize and remain affixed to vaginal epithelial cells. These unique strains can then be recovered from the vaginal milieu over time. Such sustained colonization and viability of the Lactobacillus strains is largely attributable to the novel preservation matrix disclosed herein. The protection afforded by the preservation matrix of the present invention allows the Lactobacillus strains to adhere to vaginal epithelial cells in a metabolically inactive state and retain placement while returning to an active state capable of producing functional inhibitory by-products. Such capabilities of a matrix for microbial cells has been heretofore unobserved. The preservation matrix of the present invention also provides the flexibility to allow various drying methods in the production of a commercial suppository product. One embodiment of the present invention relates to a vaginal medicant. Such a medicant includes a substantially pure bacterial culture of an isolated strain of the genus Lactobacillus having identifying characteristics which include (i) a percent vaginal epithelial cell (VEC) cohesion value (as defined below) of at least about 50% and (ii) an ability to produce greater than about 0.5 ppm of H2O2 under effective culture conditions. The vaginal medicant of the present invention also includes a preservation matrix, which contains and preserves the bacterial culture. Such a matrix includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material. In one embodiment, the matrix has a pH of from about 5.0 to about 7.0. In another embodiment, the matrix has a pH of about 7.0. The preservation matrix of the present invention is capable of maintaining at least about 106 viable, genetically stable cells for a period of at least about 12 months in vitro. In further embodiments, the matrix maintains at least about 107 viable cells for a period of at least about 12 months in vitro, and more preferably, at least about 108 viable cells for a period of at least about 12 months in vitro.
In other embodiments, the isolated Lactobacillus strain has a percent VEC cohesion value of at least about 65%, and more preferably, at least about 80% and even more preferably, at least about 95%. In still other embodiments, the isolated strain has an ability to produce at least about 10 ppm of H2O2, and more preferably, at least about 20 ppm of H2O2.
As mentioned above, one embodiment of the present invention relates to a strain of Lactobacillus as described above which adheres to vaginal epithelial cells when the strain is in a metabolically inactive state. In another embodiment, the isolated strain sustains colonization of vaginal epithelial cells in vivo for at least about 1 month. In yet another embodiment, the isolated strain maintains genetic stability in vivo for at least about 24 months. A single cell of the isolated strain is from about 1 micron to about 2 microns in width and from about 2 microns to about 4 microns in length and produces at least about 0.75 mg/100 ml of lactic acid under effective culture conditions.
In one embodiment, the Lactobacillus strain of the present invention is of the species Lactobacillus crispatus; in another embodiment, the Lactobacillus strain of the present invention is of the species Lactobacillus jensenii. In yet another embodiment, a Lactobacillus strain of the present invention has all of the identifying characteristics of Lactobacillus crispatus CTV-05, the preferred Lactobacillus strain of the present invention.
The preservation matrix of the present invention maintains at least about 106 viable cells for a period of at least about 12 months in vitro at a temperature from about 4xc2x0 C. to about 6xc2x0 C., and preferably at room temperature. In another embodiment, the matrix maintains at least about 106 viable cells for a period of at least about 18 months in vitro, and preferably at least about 24 months in vitro.
The vaginal medicant of the present invention can comprise an inert carrier including, but not limited to, maltodextrin beads or a gelatin capsule. In one embodiment, a gastrointestinal medicant includes (a) a substantially pure bacterial culture of an isolated strain of the genus Lactobacillus having identifying characteristics which include (i) a percent vaginal epithelial cell (VEC) cohesion value (as defined below) of at least about 50% and (ii) an ability to produce greater than about 0.5 ppm of H2O2 under effective culture conditions; and (b) a preservation matrix, which includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material. Such a matrix maintains at least about 106 viable, substantially pure and genetically stable cells for a period of at least about 12 months in vitro.
Another embodiment of the present invention relates to a preservation matrix for preserving microbial cells. Such a matrix includes (a) a biologically active binding agent which can include water soluble gum, carboxymethyl cellulose and gelatin; (b) an antioxidant (preferably sodium ascorbate); (c) a polyol which can include xylitol, adonitol, glycerol, dulcitol, inositol, mannitol, sorbitol and arabitol; (d) a carbohydrate which can include dextrose, lactose, maltose, sucrose, fructose, and other monosaccharides, disaccharides and oligosaccharides; and (e) a proteinaceous material which can include skim milk and albumin. Such a matrix maintains at least about 106 viable, substantially pure and genetically stable microbial cells for a period of at least about 12 months in vitro.
In another embodiment, the biologically active binding agent is at least about 10% of the total matrix by weight, the antioxidant is at least about 0.1% of the total matrix by weight, the polyol is at least about 1% of the total matrix by weight, the carbohydrate is at least about 0.5% of the total matrix by weight and the proteinaceous material is at least about 0.5% of the total matrix by weight.
In yet another embodiment, the biologically active binding agent is at least about 14% of the total matrix by weight, the antioxidant is at least about 0.5% of the total matrix by weight, the said polyol is at least about 6% of the total matrix by weight, the carbohydrate is at least about 2.5% of the total matrix by weight and the proteinaceous material is at least about 1.5% of the total matrix by weight.
Another embodiment of the present invention is a preservation matrix for preserving microbial cells which includes about 14% gelatin, about 0.5% sodium ascorbate, about 2.5% dextrose, about 1.5% skim milk and about 6% xylitol, by weight of the preservation matrix.
Yet another embodiment of the present invention relates to a method to prepare a preservation matrix for the preservation of microbial cells. Such a method includes the steps of (a) providing components which include: (i) a sterile biologically active binding agent, which can include water soluble gum, carboxymethyl cellulose and gelatin; (ii) a sterile proteinaceous material which can include skim milk and albumin; (iii) a sterile polyol which can include xylitol, adonitol, glycerol, dulcitol, inositol, mannitol, sorbitol and arabitol; (iv) a sterile antioxidant; (v) a sterile carbohydrate which can include dextrose, lactose, maltose, sucrose, fructose, and other monosaccharides, other disaccharides and other oligosaccharides; and (vi) water; and (b) mixing the components together to form a solution. The biologically active binding agent can be provided as a liquid.
Yet another embodiment of the present invention relates to a method to preserve microbial cells within a preservation matrix. Such a method includes the steps of (a) suspending a culture of at least about 106 microbial cells in a preservation matrix to form a cell matrix suspension; (b) adding the cell matrix suspension to an inert carrier to form a delivery composition; and removing water from the delivery composition. The preservation matrix includes the components as described above. In one embodiment, the method further includes the step of placing the delivery composition into a package which protects the delivery composition from moisture and oxygen during transport and storage.
In one embodiment, the inert carrier can include maltodextrin beads, and the method includes the steps of coating the cell matrix suspension onto the beads and removing water by drying the beads by fluid bed drying.
In another embodiment, the inert carrier includes a gelatin capsule, and the method includes the steps of chilling the gelatin capsule until the cell suspension matrix forms a non-fluid matrix, followed by desiccating the gelatin capsule in a desiccation chamber. The step of desiccating includes providing dry air to the desiccation chamber containing less than about 25 moisture, and more preferably, less than about 15% moisture, and even more preferably, less than about 5% moisture, at a temperature from about 24xc2x0 C. to about 32xc2x0 C. and removing humidified air from the chamber. The desiccation chamber can include a compressor, at least one hydrocarbon scrubbing filter and a chilled air compressor. The desiccation chamber can further include a desiccant column.
Yet another embodiment of the present invention relates to a method to protect a female from a vaginal infection. The method includes the step of administering to a female a vaginal medicant of the present invention as previously described herein. Another embodiment relates to a method to treat a vaginal infection, which includes the step of administering to a female a vaginal medicant as previously described herein. Vaginal infections for which such a medicant is useful include bacterial vaginosis, symptomatic yeast vaginitis, gonorrhea, chlamydia, trichomoniasis, human immunodeficiency virus infection, urinary tract infection or pelvic inflammatory disease.
Yet another embodiment of the present invention relates to a method to reduce the risk of infection of a human by human immunodeficiency virus (HIV), which includes the step of administering to a human a medicant of the present invention as previously described herein. Administration of such a medicant can reduce the risk of HIV infection of a human by at least about two-fold.
Another embodiment of the present invention relates to a method to prevent infection of a human by symptomatic yeast vaginitis, which includes administering to a human a vaginal medicant of the present invention as previously described herein. In a preferred embodiment, the Lactobacillus strain included in the medicant is Lactobacillus crispatus CTV-05.
Another embodiment of the present invention relates to a method to prevent infection of a human by a gastrointestinal infection, which includes administering to a human a vaginal medicant of the present invention as described herein.
Yet another embodiment of the present invention is a method to prevent preterm birth, which includes administering to a pregnant female a vaginal medicant of the present invention as described herein.
One embodiment of the present invention is a method to enhance metabolism of estrogen in the vagina and bowel, which includes administering to a female a vaginal medicant of the present invention as described herein.
Yet another embodiment of the present invention relates to a vaginal medicant which includes (a) a substantially pure bacterial culture of at least two isolated strains of the genus Lactobacillus having identifying characteristics which include (i) a percent vaginal epithelial cell (VEC) cohesion value (as defined below) of at least about 50% and (ii) an ability to produce greater than about 0.5 ppm of H2O2 under effective culture conditions; and (b) a preservation matrix, which includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material. Such a matrix maintains at least about 106 viable, substantially pure and genetically stable cells for a period of at least about 12 months in vitro. Such a medicant can be used to treat at least two vaginal infections which include bacterial vaginosis, symptomatic yeast vaginitis, gonorrhea, chlamydia, trichomoniasis, human immunodeficiency virus infection, urinary tract infection or pelvic inflammatory disease. In one embodiment, the bacterial culture includes a first strain which is effective for treating a first vaginal infection and a second strain which is effective for treating a second vaginal infection. In a preferred embodiment, the first strain is Lactobacillus crispatus CTV-05 and the second strain is of the species Lactobacillus jensenii. 
Another embodiment of the present invention relates to a vaginal medicant which includes (a) a substantially pure bacterial culture of isolated Lactobacillus crispatus CTV-05; and (b) a preservation matrix which includes about 14% gelatin, about 0.5% sodium ascorbate, about 2.5 dextrose, about 1.5% skim milk and about 6% xylitol. The matrix maintains at least about 106 viable, genetically stable cells for a period of at least about 12 months in vitro, and preserves desirable characteristics of the Lactobacillus crispatus CTV-05. Such characteristics include an ability to adhere to vaginal epithelial cells in a metabolically inactive state, an ability to produce greater than about 0.5 ppm of H2O2 under effective culture conditions, and a percent vaginal epithelial cell (VEC) cohesion value of at least about 50%.